The goal of this research is to determine molecular rules and driving forces that regulate biological functions of protein complexes. Human hemoglobin (Hb) is being studied to understand (i) the intermediate states and pathways whereby subunit interactions generate physiological cooperativity, and (ii) how mechanisms of the intermediates are modulated by physiological effectors (H+, CO 2, C 1-). This program includes: (a) development of new methods to analyze energetic contributions to cooperativity by the intermediate tetamers; (b) correlation of the resulting site-specific energetics with accompanying structure changes; c usage of single amino acid modifications to probe structural elements which mediate the energetic components of Hb cooperativity. Our earlier work found that the Hb structural switches follow specific combinatorial rules: studies with single amino acid modifications to the binding intermediates have revealed important symmetry/asymmetry features of the Hb tetramer's structural energetics . We plan to extend these studies using recombinant Hb's with designed combinations of altered residue sites for the O2 intermediates. Subunit interactions of the recombinant systems will be studied by techniques of hybridization, assembly and kinetics, as well as by direct O2 binding. Structural features will be analyzed by X-ray crystallography. The Hb regulatory system is an important prototype for a large family of cooperative multi-site regulatory assemblies. Thus the methods developed by this program should have wide applicability. Deeper understanding of human Hb mechanisms is also of current interest to the potential design of red cell substitute oxygen carriers and drug delivery systems.